RU2708992C2 - Allele-specific amplification using composition of overlapping oligonucleotides as non-allele-specific primer and allele-specific blocker - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to the biotechnology. Described is a composition of oligonucleotides for amplification of a target sequence. Composition contains an oligonucleotide as a blocker, including a functional group or an uncomplementary section of sequence at 3'-end or nearby it, which prevents enzymatic elongation. Oligonucleotide as a blocker includes a first sequence comprising a neutral to the target sub-sequence and a blocking variable subsequence, where the blocking variable subsequence is flanked at its 3'- and 5'-ends of a subsequence which is neutral with respect to the target and is inextricable with a neutral to the target of the sub-sequence. Oligonucleotide as the first primer is sufficient to induce enzymatic elongation. Oligonucleotide as a first primer includes a second sequence, where the second sequence is overlapped with a neutral to the target subsequence of the oligonucleotide as a blocker for at least 5 nucleotides, so that the second sequence includes an overlapping sub-sequence and a nonoverlapping sub-sequence, and the second sequence does not include a blocking variable subsequence, second sequence creates standard free hybridisation energy (ΔG^ο _PT), and the first sequence generates standard free hybridisation energy (ΔG^ο _BT), which satisfies the following condition: +2 kcal/mol ≥ΔG^ο _PT-ΔG^ο _BT≥-8 kcal/mol. Concentration of the oligonucleotide as a blocker is approximately 2 - approximately 10,000 times higher than the concentration of the oligonucleotide as the first primer.

EFFECT: invention extends range of means for amplification.

47 cl, 14 dwg, 1 tbl, 11 ex

Description

Cross reference to related applications

This application claims the priority of provisional patent application US No. 62/000114, filed May 19, 2014, the description of which is incorporated here by reference in full.

Sequence List

A list of sequences is provided with this application only in electronic form and is incorporated herein by reference. The sequence listing text file “14-21013-WO (260947,00251) _SL.txt” was created on May 18, 2015 and is 13,144 bytes.

BACKGROUND OF THE INVENTION

Small differences in the sequence of DNA and RNA can lead to large differences in physical health in general and the well-being of organisms, including humans. For example, replacing one base in the bacterial genome can lead to antibiotic resistance, and replacing one base in the human genome can lead to cancer progression. Due to the achievement of the full development of the field of genomics and the concurrent discovery of many sequences and molecules of nucleic acid biomarkers, there is a great need on the part of the biotechnology industry to develop reliable, resistant to abnormal, inexpensive and accurate nucleic acid analyzes that can distinguish single base substitutions. In particular, many PCR-based assays have been developed that are allele-specific.

PCR-based approaches to the selective detection of rare mutations can be broadly divided into two series of approaches: (A) allele-specific primers and (B) non-allele-specific primers with allele-specific blockers.

Examples of allele-specific primers include an amplification system for mutation identification (ARMS), PCR using allele-specific blockers (ASB-PCR), and competitive allele-specific TaqMan PCR (castPCR). Examples (B) include PCR using PNA blockers and PCR using co-amplification at lower temperature denaturation (COLD-PCR).

Prior to the present invention, allele-specific primers were generally better (more susceptible to mutations) when detecting known mutations of the same base, while non-allele-specific primers with allele-specific blockers are typically used to detect multiple closely grouped mutations (hot spots) or unknown sequences with mutations.

Allele-specific primers for PCR, such as ARMS, ASB-PCR, castPCR, as a rule, have an allele-specific nucleotide at the extreme 3 ′ position of the primer. In the case of allele-specific PCR primers, the distinctive ability of the DNA polymerase enzyme to specific extension of correctly paired bases is used, but with the restriction that when an event of incorrect extension by DNA polymerase occurs, the rare allelic nucleotide that was part of the primer becomes incorporated into the matrix, and subsequently amplification cycles become non-specific. Allele-specific PCR primers are specific only up to the first improper amplification event. Allele-specific detection using non-allele-specific primers requires precise control of the denaturation temperature and restriction analysis of small sequences. Allele-specific detection using non-allele-specific primers has low sensitivity to mutations and is more vulnerable to polymerase-introduced errors.

Thus, an oligonucleotide composition is required as a non-allele-specific primer and an allele-specific blocker with increased allele-specific amplification and increased sensitivity to mutations. Therefore, the development of new approaches suitable for detecting a small amount of a target in a large excess of variant is a prerequisite for diagnostic applications. Thus, the present invention provides a composition of oligonucleotides as a non-allele-specific primer and an allele-specific blocker to provide a more accurate and effective means for applications in the form of diagnosing diseases such as cancer diagnosis and the like.

Summary of the invention

The present invention relates to an overlapping structure of an oligonucleotide as a non-allele-specific primer and an oligonucleotide as an allele-specific blocker for allele-specific amplification.

The present invention provides an oligonucleotide composition comprising a blocker and an oligonucleotide as a first primer. The oligonucleotide as a blocker includes a first sequence containing a neutral sequence with respect to the target and a blocking variable subsequence. However, in some cases, the oligonucleotide as a blocker may not include a blocking variable subsequence if the detectable target nucleic acid is intended to detect an insert. The blocking variable subsequence is flanked at its 3'- and 5'-ends with a neutral subsequence in relation to the target and is inextricable with a neutral subsequence in relation to the target. The oligonucleotide as the first primer is sufficient to induce enzymatic extension; as used herein, an oligonucleotide comprises a second sequence as a first primer. The second sequence overlaps with the 5'-end of the target-neutral subsequence for at least 5 nucleotides, so that the second sequence includes an overlapping subsequence and a non-overlapping subsequence. The second sequence does not include any sequence homologous to the blocking variable subsequence.

The present invention also relates to a method for amplifying a target sequence. The method includes obtaining a sample containing one or more copies of a first nucleic acid having a variant sequence, and at least one copy of a second nucleic acid; as used herein, the second nucleic acid has a target sequence. Each of the target sequence and variant sequence includes a homologous subsequence and a variable subsequence. The variable subsequence also includes at least one nucleotide. In addition, the variable subsequence of a target sequence is a target-specific subsequence, and the variable subsequence of a variant sequence is not a target-specific subsequence.

After receiving the sample, the oligonucleotide as a blocker is introduced into the sample. The oligonucleotide as a blocker includes a first sequence containing a neutral sequence with respect to the target and a blocking variable subsequence. The target-neutral subsequence is complementary to the part of the homologous subsequence, and the blocking variable subsequence is complementary to the non-target-specific subsequence. In addition, the blocking variable subsequence is flanked at its 3'- and 5'-ends with a target-neutral subsequence and is inextricable with a target-neutral subsequence.

After that, the oligonucleotide as the first primer is introduced into the sample. The oligonucleotide as the first primer is sufficient to induce enzymatic extension. The oligonucleotide as the first primer includes a second sequence that is complementary to the second part of the homologous subsequence. In addition, the second sequence overlaps with the 5'-end of the target-neutral subsequence for at least 5 nucleotides, so that the second sequence includes an overlapping subsequence and non-overlapping subsequence. The second sequence does not include any sequence complementary to the variable subsequence.

In the next step, DNA polymerase, nucleoside triphosphates and one or more reagents necessary for the polymerase amplification of nucleic acids are introduced into the sample; and cause a reaction in the sample under conditions sufficient to achieve amplification of nucleic acids.

An advantage of the present invention are approaches corresponding to allele-specific primers and / or exceeding them, with increased sensitivity to mutations.

Another advantage of the present invention is a simple two-step thermal cycle protocol with considerable resistance to temperature excursions (8 ^ο C).

Another advantage of the present invention is the provision of inexpensive reagents in the form of DNA primers and blockers without complex modifications of the backbones or nucleotides.

Another advantage of the present invention is compatibility with high-quality enzymes.

A further advantage of the present invention is that the allele-specific blocker binds more strongly to the variant than to the target, so that the non-allele-specific primer more successfully displaces the blockers associated with the target compared to the blockers associated with the variant. The advantage of using non-allele-specific primers is that falsely amplified variants result in amplification products (amplicons) carrying a variant sequence, rather than a target sequence. Thus, specificity is exacerbated at each cycle.

This summary is provided to precede the disclosure, present some aspects, advantages and new features of the invention in a simplified form, which are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

Brief Description of the Drawings

The above summary, as well as the following detailed description of illustrative embodiments, will be better understood when reading along with the accompanying drawings. For the purpose of illustrating the present invention, exemplary constructions of the present invention are shown in the drawings. However, the invention is not limited to the specific methods and means described herein.

In FIG. 1 shows a schematic view of a non-allele-specific primer and allele-specific blocker for allele-specific amplification in accordance with the present invention.

In FIG. 2 shows in schematic form the thermodynamic development of a primer and blocker in accordance with the present invention.

In FIG. 3 shows in a schematic form the calculation of ΔG ^ο values based on the sequence in accordance with the present invention. FIG. 3 discloses SEQ ID NO: 1, 33, 1, 34, 2, 33, 2, and 34, respectively, in order of appearance.

In FIG. 4 is a schematic representation of the detection of a rare allele in human genomic DNA in accordance with Example 1. FIG. 4 discloses SEQ ID NO: 3-4, 35, 32, 36, and 32, respectively, in order of appearance.

In FIG. 5 is a schematic representation of resistance to rare temperature deviations in accordance with Example 2. FIG. 5 discloses SEQ ID NO: 5-6 and 37-38, respectively, in order of appearance.

In FIG. 6A shows in diagrammatic form the increased flexibility of primer design in accordance with Example 3.

In FIG. 6B are a schematic representation of a nucleic acid sequence of 4 sets of primer and blocker pairs that amplify human SNP rs3789806 in accordance with Example 3. FIG. 6B discloses SEQ ID NOs: 5-12 and 39-40, respectively, in order of appearance.

In FIG. 7A is shown in a schematic view of the simulation of effects ΔG ^ο _rxn1 with a fixed ΔΔG ^ο = 2 kcal / mol in accordance with Example 4.

In FIG. 7B (I) is a schematic representation of an experimental test on the rs4939827 SMAD7 in accordance with Example 4. FIG. 7B (I) discloses SEQ ID NO: 3, 13-16, 4, 17, and 35-36, respectively, in order of appearance.

In FIG. 7B (II) is a schematic representation of an experimental test on the rs4939827 SMAD7 in accordance with Example 4. FIG. 7B (II) discloses SEQ ID NO: 3, 18-23, 36, and 35, respectively, in order of appearance.

In FIG. 8 is a schematic illustration of a blocker with a 3 ′ non-homologous portion in accordance with Example 5. FIG. 8 discloses SEQ ID NO: 5, 24, and 37-38, respectively, in order of appearance.

In FIG. 9A is a schematic representation of multiplexing using TaqMan® probes in accordance with Example 6.

In FIG. 9B is a schematic representation of the multiplexing using TaqMan® probes in accordance with Example 6.

In FIG. 10 shows in schematic form the protectors for the primer and blocker in accordance with Example 7.

In FIG. 11A is a schematic illustration of the detection of a deletion (rs200841330 STAG2) in accordance with Example 8. FIG. 11A discloses SEQ ID NOs: 25-26 and 41-42, respectively, in order of appearance.

In FIG. 11B is a schematic view of the detection of an insert (rs200841330 STAG2) in accordance with Example 8. FIG. 11B discloses SEQ ID NO: 25, 43, 42, and 41, respectively, in order of appearance.

In FIG. 12 is a schematic illustration of a 5ʹ blocker in accordance with Example 9. FIG. 12 discloses SEQ ID NOs: 27-28 and 44-45, respectively, in order of appearance.

In FIG. 13 shows in schematic form the specific amplification of DNA encoding a fungal ribosomal RNA in accordance with Example 10.

In FIG. 14 is a schematic diagram of a Dual NASP in accordance with Example 11. FIG. 14 discloses SEQ ID NO: 29, 10, 46-49, and 30-31, respectively, in order of appearance.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described with reference to specific embodiments and with reference to some drawings, but the present invention is not limited thereto, but only by the claims. The presented drawings are only diagrams and are not limiting. In the drawings, the dimensions of some elements may be oversized or distorted and not scaled for illustrative purposes. Locally, elements of the invention are designated as “a” or “an” at the first cast and are designated as “the” or “indicated” in the case of the second or subsequent casts, if anything else is not specified.

And the present invention will be described in more detail here later with reference to the accompanying drawings, in which some, but not all, embodiments of the present invention are shown. Indeed, the present invention can be implemented in many different forms and should not be construed as limited by the embodiments set forth herein, rather, these embodiments are provided so that this description meets all legal requirements.

Definition

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art.

As used herein, the term “oligonucleotide as a blocker” refers to at least one continuous chain of from about 12 to about 100 nucleotides in length and, if indicated here, may also include a functional group or nucleotide sequence at its 3'-end, which prevents enzymatic elongation during an amplification process such as a polymerase chain reaction.

As used herein, the term “primer oligonucleotide” refers to a molecule comprising at least one continuous chain of from about 12 to about 100 nucleotides in length and sufficient to provide enzymatic extension during an amplification process, such as a polymerase chain reaction.

As used herein, the term “target neutral sequence” refers to a nucleotide sequence that is complementary to the sequence in both the target nucleic acid and the nucleic acid variant. For example, a desired nucleic acid sequence that is a target for amplification (target nucleic acid) may exist in a sample with a nucleic acid molecule having a substantially homologous sequence with a target nucleic acid, except for the variable region (nucleic acid variant), however, such a variable region in some cases differs only in one nucleotide from the target nucleic acid. In this example, a target-neutral subsequence is complementary to at least a portion of the homologous sequence common to two nucleic acids, but not to the variable region. Thus, the term “blocking variable subsequence” as used herein refers to the nucleotide sequence of an oligonucleotide as a blocker that is complementary to the variable region of a nucleic acid variant.

As used herein, the term “overlapping subsequence” refers to a nucleotide sequence of at least 5 nucleotides of an oligonucleotide as a primer that is homologous to a portion of the oligonucleotide sequence as a blocker used in the composition described herein. The overlapping subsequence of the oligonucleotide as a primer can be homologous to any part of the neutral to the target subsequence of the oligonucleotide as a blocker, or 5 ′ or 3 ′ of the blocking variable subsequence. Thus, the term “non-overlapping subsequence” refers to an oligonucleotide sequence as a primer that is not an overlapping subsequence.

As used herein, the term “target sequence” refers to a nucleotide sequence of a nucleic acid that carries the desired allele, such as a single nucleotide polymorphism, which must be amplified, identified, or otherwise isolated. As used herein, the term “sequence variant” refers to a nucleotide sequence of a nucleic acid that does not carry the desired allele. For example, in some cases, the variant variant carries the wild-type allele, while the target sequence carries the mutant allele. Thus, in some cases, the sequence variant and the target sequence come from a common locus in the genome, so that each of the sequences can be essentially homologous except for the region carrying the desired allele, nucleotide or group of nucleotides that differs between the two sequences.

The present invention relates to a composition of oligonucleotides. In one embodiment of the present invention, the oligonucleotide composition comprises an oligonucleotide as a non-allele-specific primer and an oligonucleotide as an allele-specific blocker for allele-specific amplification.

In another embodiment of the present invention, the oligonucleotide composition comprises an oligonucleotide as a blocker and an oligonucleotide as a first primer. The oligonucleotide as a blocker includes a first sequence containing a neutral sequence with respect to the target and a blocking variable subsequence. The blocking variable subsequence is flanked at its 3'- and 5'-ends with a neutral subsequence in relation to the target and is inextricable with a neutral subsequence in relation to the target. In other words, a blocking variable subsequence can divide a subsequence neutral with respect to the target into two parts, a part located 3 ′ from the blocking variable subsequence and a part located 5 ′ from it. The oligonucleotide as the first primer is sufficient to induce enzymatic extension; as used herein, an oligonucleotide comprises a second sequence as a first primer. The second sequence overlaps with a target-neutral subsequence of at least 5 nucleotides, so the second sequence includes an overlapping subsequence and a non-overlapping subsequence. The second sequence does not include a blocking variable subsequence. Thus, an oligonucleotide as a primer can be characterized as a non-allele-specific primer. The overlapping region may be homologous to the part of the oligonucleotide subsequence, neutral with respect to the target, as a blocker, either from the 5'-side of the blocking variable subsequence or from the 3'-side from the blocking variable subsequence. As shown in FIG. 1, the overlapping subsequence of the oligonucleotide as a primer is homologous to the target of the blocker subsequence on the 5 ′ side of the blocking variable subsequence (designated as “C” on the blocker). Here, the target-neutral subsequence is parts of the blocker on either side of the variable blocking subsequence C. In FIG. 12 shows one example in which the overlapping subsequence of the primer is homologous to the part of the blocker subsequence that is neutral with respect to the target, which is 3 ′ from the blocking variable subsequence C.

In yet another embodiment of the present invention, the oligonucleotide as a blocker further comprises a functional group or non-complementary portion of a sequence at or near the 3'-end that prevents enzymatic extension. In one case, the functional group of the oligonucleotide as a blocker is selected, but not limited to, from the group consisting of a 3-carbon spacer or dideoxynucleotide.

The second sequence creates the standard free hybridization energy (ΔG ^ο _PT ), and the first sequence creates the standard free hybridization energy (ΔG ^ο _BT ), which satisfies the following condition:

+2 kcal / mol≥ΔG ^ο _PT -ΔG ^ο _BT ≥-8 kcal / mol

A non-overlapping subsequence creates a standard free hybridization energy (ΔG ^ο ₃ ) that satisfies the following condition:

-4 kcal / mol≥ΔG ^ο ₃ ≥-12 kcal / mol

In one case, the second sequence overlaps with the 5'-end of the subsequence, neutral with respect to the target, from about 5 nucleotides to about 40 nucleotides. In another case, the second sequence overlaps with the 5'-end of the subsequence, neutral with respect to the target, from about 7 nucleotides to about 30 nucleotides.

In one case, the concentration of the oligonucleotide as a blocker is about 2 to about 10,000 times higher than the concentration of the oligonucleotide as the first primer. In another case, the concentration of the oligonucleotide as a blocker is about 5 to about 1000 times higher than the concentration of the oligonucleotide as the first primer.

In yet another embodiment of the present invention, the oligonucleotide composition further comprises an oligonucleotide as a second primer sufficient to induce enzymatic extension. The oligonucleotide as the second primer includes a third sequence that does not overlap with the first sequence or the second sequence, is neutral with respect to the target and sufficient for use with the oligonucleotide as the first primer to amplify a nucleic acid region using a polymerase chain reaction.

In yet another embodiment of the present invention, the oligonucleotide composition further comprises an oligonucleotide as a second blocker. The oligonucleotide as a second blocker comprises a fourth sequence comprising a second target neutral sequence and a second variable blocking subsequence. The second blocking variable subsequence has the same sequence as the non-target-specific subsequence of the target nucleic acid, given that it is usually designed to bind the antisense sequence of the target. The second blocking variable subsequence is flanked at its 3'- and 5'-ends with a second target-neutral subsequence and is inextricable with the second target-neutral subsequence. The third sequence overlaps with the 5'-end of the second target-neutral subsequence for at least 5 nucleotides, so that the third sequence includes a second overlapping subsequence and a second non-overlapping subsequence.

In yet another embodiment of the present invention, the oligonucleotide as a second blocker further comprises a second functional group or a second non-complementary portion of the sequence at or near the 3 ′ end, which prevents enzymatic extension. In one case, the second functional group of the oligonucleotide as the second blocker is selected, but not limited to, from the group consisting of a 3-carbon spacer or dideoxynucleotide.

The third sequence creates the standard free hybridization energy (ΔG ^ο _PT2 ), and the fourth sequence creates the standard free hybridization energy (ΔG ^ο _BT2 ), which satisfies the following condition:

+2 kcal / mol≥ΔG ^ο _{PT2 -ΔG} ^ο _BT2 ≥-8 kcal / mol

The second non-overlapping subsequence creates the standard free hybridization energy (ΔG ^ο ₆ ), which satisfies the following condition:

-4 kcal / mol≥ΔG ^ο ₆ ≥-12 kcal / mol

In one case, the third sequence overlaps with the 5'-end of the second target-neutral subsequence for from about 5 nucleotides to about 40 nucleotides. In another case, the third sequence overlaps with the 5'-end of the second target-neutral subsequence from about 7 nucleotides to about 30 nucleotides.

In any of the above embodiments, the length of each of the oligonucleotide as a primer and the oligonucleotide as a blocker may be from about 12 nucleotides to about 100 nucleotides. In any of the above embodiments, the length of each of the oligonucleotide as a primer and the oligonucleotide as a blocker may be from about 15 nucleotides to about 90 nucleotides. In any of the above embodiments, the length of each of the oligonucleotide as a primer and the oligonucleotide as a blocker may be from about 20 nucleotides to about 80 nucleotides. In any of the above embodiments, the length of each of the oligonucleotide as a primer and the oligonucleotide as a blocker may be from about 20 nucleotides to about 70 nucleotides. In any of the above embodiments, the length of each of the oligonucleotide as a primer and the oligonucleotide as a blocker may be from about 20 nucleotides to about 60 nucleotides. In any of the above embodiments, the length of each of the oligonucleotide as a primer and the oligonucleotide as a blocker may be 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

In one case, the concentration of the oligonucleotide as the second blocker is about 2 to about 10,000 times higher than the concentration of the oligonucleotide as the second primer. In another case, the concentration of the oligonucleotide as the second blocker is about 5 to about 1000 times higher than the concentration of the oligonucleotide as the second primer. In another case, the concentration of the oligonucleotide as the second blocker is about 10 to about 900 times higher than the concentration of the oligonucleotide as the second primer. In another case, the concentration of the oligonucleotide as the second blocker is about 20 to about 800 times higher than the concentration of the oligonucleotide as the second primer. In another case, the concentration of the oligonucleotide as the second blocker is about 30 to about 700 times higher than the concentration of the oligonucleotide as the second primer. In another case, the concentration of the oligonucleotide as the second blocker is about 40 to about 600 times higher than the concentration of the oligonucleotide as the second primer. In another case, the concentration of the oligonucleotide as the second blocker is about 50 to about 500 times higher than the concentration of the oligonucleotide as the second primer. In another case, the concentration of the oligonucleotide as the second blocker is about 60 to about 400 times higher than the concentration of the oligonucleotide as the second primer. In another case, the concentration of the oligonucleotide as the second blocker is about 70 to about 300 times higher than the concentration of the oligonucleotide as the second primer. In another case, the concentration of the oligonucleotide as the second blocker is about 80 to about 200 times higher than the concentration of the oligonucleotide as the second primer. In another case, the concentration of the oligonucleotide as the second blocker is about 90 to about 100 times higher than the concentration of the oligonucleotide as the second primer.

In yet another embodiment of the present invention, the oligonucleotide composition further comprises a reagent necessary for the polymerase chain reaction.

In yet another embodiment of the present invention, the oligonucleotide composition further comprises a plurality of nucleoside triphosphates.

In yet another embodiment of the present invention, the oligonucleotide composition further comprises DNA polymerase or RNA polymerase.

In yet another embodiment of the present invention, the oligonucleotide composition further comprises a reagent necessary for the polymerase chain reaction, a plurality of nucleoside triphosphates, DNA polymerase.

In yet another embodiment of the present invention, the blocking variable subsequence is a single nucleotide.

The present invention also relates to a method for amplifying a target sequence. The method includes the steps of (a) obtaining a sample containing one or more copies of a first nucleic acid having a sequence variant, and at least one copy of a second nucleic acid; as used herein, the second nucleic acid has a target sequence. Each of the target sequence and sequence variant has a homologous subsequence and a variable subsequence. The variable subsequence also includes at least one nucleotide, but may include 2-5 nucleotides. In addition, the variable subsequence of the target sequence is a target-specific subsequence, and the variable subsequence of a variant sequence is not a target-specific subsequence.

The method further comprises the step of (b) introducing the oligonucleotide as a blocker in the sample; in this description, the oligonucleotide as a blocker includes a first sequence containing a neutral sequence with respect to the target and a blocking variable subsequence. The target-neutral subsequence is complementary to the part of the homologous subsequence, and the blocking variable subsequence is complementary to the non-target-specific subsequence. In addition, the blocking variable subsequence is flanked at its 3'- and 5'-ends with a target-neutral subsequence and is inextricable with a target-neutral subsequence.

The method further comprises the step of (c) introducing the oligonucleotide as the first primer into the sample. The oligonucleotide as the first primer is sufficient to induce enzymatic extension. The oligonucleotide as the first primer includes a second sequence. In addition, the second sequence is complementary to the second part of the homologous subsequence and can overlap with the 5'-end of the target-neutral subsequence for at least 5 nucleotides, so that the second sequence includes an overlapping subsequence and non-overlapping subsequence. The second sequence does not include any sequence complementary to the variable subsequence.

The method further comprises the step of (d) introducing into the sample DNA polymerase, nucleoside triphosphates and one or more reagents necessary for the polymerase amplification of nucleic acids; and then (e) inducing a reaction in the sample under conditions sufficient to achieve amplification of nucleic acids.

In one embodiment of the present invention, the oligonucleotide as a blocker further comprises a functional group or non-complementary portion of a sequence at or near the 3 ′ end that prevents enzymatic extension. In one case, the functional group is selected, but not limited to, from the group consisting of a 3-carbon spacer or dideoxynucleotide.

The second sequence creates the standard free hybridization energy (ΔG ^ο _PT ), and the first sequence creates the standard free hybridization energy (ΔG ^ο _BT ), which satisfies the following condition:

+2 kcal / mol≥ΔG ^ο _PT -ΔG ^ο _BT ≥-8 kcal / mol

A non-overlapping subsequence creates a standard free hybridization energy (ΔG ^ο ₃ ) that satisfies the following condition:

-4 kcal / mol≥ΔG ^ο ₃ ≥-12 kcal / mol

In one case, the second sequence overlaps with the 5'-end of the blocker subsequence, neutral with respect to the target, from about 5 nucleotides to about 40 nucleotides (ie, it is an “overlapping subsequence”). In other words, the second sequence includes an overlapping subsequence that is homologous to 5 nucleotides — approximately 40 nucleotides neutral with respect to the target of the blocker subsequence, which is located on the 5′-side of the blocker variable sequence of the blocker. In another case, the second sequence overlaps with the 5'-end of the subsequence, neutral with respect to the target, from about 7 nucleotides to about 30 nucleotides. In yet another case, the second sequence includes an overlapping subsequence that is homologous to the neutral part with respect to the target of the blocker subsequence, which is located on the 3′-side of the blocker variable subsequence of the blocker.

In one case, the concentration of the oligonucleotide as a blocker introduced into the sample is about 2 to about 10,000 times higher than the concentration of the oligonucleotide as the first primer introduced into the sample. In another case, the concentration of the oligonucleotide as a blocker introduced into the sample is about 5 to about 1000 times higher than the concentration of the oligonucleotide as the first primer introduced into the sample. In another case, the concentration of the oligonucleotide as a blocker introduced into the sample is about 10 to about 500 times higher than the concentration of the oligonucleotide as the first primer introduced into the sample. In another case, the concentration of the oligonucleotide as a blocker introduced into the sample is about 20 to about 250 times higher than the concentration of the oligonucleotide as the first primer introduced into the sample. In another case, the concentration of the oligonucleotide as a blocker introduced into the sample is about 40 to about 125 times higher than the concentration of the oligonucleotide as the first primer introduced into the sample. In another case, the concentration of the oligonucleotide as a blocker introduced into the sample is about 50 to about 100 times higher than the concentration of the oligonucleotide as the first primer introduced into the sample. In another case, the concentration of the oligonucleotide as a blocker introduced into the sample is about 300 to about 400 times higher than the concentration of the oligonucleotide as the first primer introduced into the sample. In another case, the concentration of the oligonucleotide as a blocker introduced into the sample is about 500 to about 600 times higher than the concentration of the oligonucleotide as the first primer introduced into the sample.

In another embodiment of the present invention, the DNA polymerase is a thermostable DNA polymerase.

In yet another embodiment of the present invention, the method further comprises conditions sufficient to achieve amplification of nucleic acids by subjecting the sample to at least 10 cycles. In each cycle are carried out for at least two different temperature exposure, one effects a temperature of at least 85 ^ο C and one impact temperature of not more than 75 ^ο C.

In yet another embodiment of the present invention, the method further comprises the step of introducing into the sample an enzyme selected from the group consisting of a single-stranded enzyme, recombinase, helicase, RNase, reverse transcriptase, or any combination thereof.

In yet another embodiment of the present invention, the method further comprises the step of introducing the oligonucleotide as a second primer into the sample. The oligonucleotide as a second primer includes a third sequence. In addition, the third sequence does not overlap with the variable subsequence. In addition, the third sequence is neutral with respect to the target and sufficient for use with the oligonucleotide as the first primer to amplify a portion of the nucleic acid containing the target sequence.

In yet another embodiment of the present invention, the method further comprises the step of introducing the oligonucleotide as a second blocker into the sample. The oligonucleotide as a second blocker comprises a fourth sequence; in this description, the fourth sequence contains a second target-neutral subsequence and a second blocking variable subsequence. In addition, the second blocking variable subsequence is a sequence complementary to the blocking variable subsequence. In addition, the second blocking variable subsequence is flanked at its 3'- and 5'-ends by a second target-neutral subsequence and is inextricable with the second target-neutral subsequence. In addition, the third sequence overlaps with the 5'-end of the second target-neutral subsequence for at least 5 nucleotides, so that the third sequence includes a second overlapping subsequence and a second non-overlapping subsequence.

In yet another embodiment of the present invention, the oligonucleotide as a second blocker further comprises a second functional group or a second non-complementary portion of the sequence at or near the 3 ′ end, which prevents enzymatic extension. In one case, the second functional group is selected, but not limited to, from the group consisting of a 3-carbon spacer or dideoxynucleotide.

The third sequence creates the standard free hybridization energy (ΔG ^ο _PT2 ), and the fourth sequence creates the standard free hybridization energy (ΔG ^ο _BT2 ), which satisfies the following condition:

+2 kcal / mol≥ΔG ^ο _{PT2 -ΔG} ^ο _BT2 ≥-8 kcal / mol

The second non-overlapping subsequence creates the standard free hybridization energy (ΔG ^ο ₆ ), which satisfies the following condition:

-4 kcal / mol≥ΔG ^ο ₆ ≥-12 kcal / mol

In one case, the third sequence overlaps with the 5'-end of the second target-neutral subsequence for from about 5 nucleotides to about 40 nucleotides. In another case, the third sequence overlaps with the 5'-end of the second target-neutral subsequence from about 7 nucleotides to about 30 nucleotides.

In one case, the concentration of the oligonucleotide as the second blocker introduced into the sample is about 2 to about 10,000 times higher than the concentration of the oligonucleotide as the second primer introduced into the sample. In another case, the concentration of the oligonucleotide as the second blocker introduced into the sample is about 5 to about 1000 times higher than the concentration of the oligonucleotide as the second primer introduced into the sample.

In yet another embodiment of the present invention, the method of amplifying a target sequence further comprises the step of extracting an aliquot from the sample and repeating steps (b) through (e) inclusive as defined above.

In FIG. 1 shows a schematic view of a non-allele-specific primer and an allele-specific blocker for allele-specific amplification. The nucleic acid reagent mixture includes two types of oligonucleotides, a primer (oligonucleotide as a primer) and a specific blocker variant (oligonucleotide as a blocker), which act together to ensure reliable amplification of the diluted target in combination with the polymerase amplification method. The blocker can be modified at the 3'-end as a result of a non-extendable modification, such as a dideoxynucleotide or a 3-carbon linker. The primer and blocker sequences are designed rationally based on the thermodynamics of their hybridization with the target nucleic acid sequence and a variant of the nucleic acid sequence. In some embodiments, the blocker is present in a significantly higher concentration than the primer, so that the vast majority of the target and nucleic acid sequence variants bind to the blocker prior to binding to the primer. The primer binds temporarily to the molecules of the blocker-target or blocker-variant and has the ability to displace the blocker when bound to the target or variant. Since the blocker sequence is specific for the target variant, its displacement from the variant is less thermodynamically advantageous than its displacement from the target. Accordingly, a non-allele-specific primer amplifies a target sequence with higher yield / efficiency than it amplifies a sequence variant. The non-allele-specific nature of the primer means that the falsely amplified version of the sequence carries the allele variant rather than the target allele, so subsequent amplification cycles also show amplification bias in favor of the target.

A high concentration of the variant-specific blocker leads to a higher probability of first binding of the blocker to both the target and the variant before the primer can bind. The primer initiates binding with both the target-blocker complex and the variant-blocker complex, using a unique site that does not overlap with the blocker, and subsequently, possibly displaces the blocker through a chain displacement process without enzyme involvement.

In FIG. 2 shows in a schematic form the thermodynamic design of a primer and blocker. The primer and blocker sequences are rationally designed to achieve the desired reaction thermodynamics. Here, the primer, blocker, target, and nucleic acid sequence variants are subdivided into regions comprising successive nucleotides denoted by the numbers 1 to 8 inclusive in FIG. 2. The standard free hybridization energy between the primer and the target (ΔG ^ο _PT ) and the standard free hybridization energy between the blocker and the target (ΔG ^ο _BT ) satisfy the condition:

+2 kcal / mol≥ΔG ^ο _PT -ΔG ^ο _BT ≥-8 kcal / mol

Systems comprising a primer and a blocker that satisfy the above inequality generate a large difference in the yields of the hybridization reaction between the primer bound to the target and the primer bound to the variant in each cycle, which leads to target-specific amplification.

The process of replacing a chain without enzyme involvement relies on the relative thermodynamics of primer binding compared to blocker binding. The blocker hybridizes more actively with the variant (with standard free binding energy ΔG ^ο _BV ) than with the target (with standard free binding energy ΔG ^ο _BT , ΔG ^ο _BT > ΔG ^ο _BV , since a more negative value ΔG ^ο indicates greater activity). In another embodiment of the present invention, the primer hybridizes equally actively with the variant (with standard free binding energy ΔG ^ο _PV ), as well as with the target (with standard free binding energy ΔG ^ο _PT , ΔG ^ο _PV = ΔG ^ο _PT ).

The blocker primer displacement reaction upon binding to the target, BT + P⌠PT + B, has a standard free energy ΔG ^ο _rxn1 = ΔG ^ο _PT -ΔG ^ο _BT , which is more negative (more favorable) than the blocker primer displacement reaction upon binding with option, BV + P⌠PV + B, which has a standard free energy ΔG ^ο _rxn2 = ΔG ^ο _PV -ΔG ^ο _BV . For the present invention to work well, the primer and blocker should usually be designed so that ΔG ^ο _rxn1 ≤0 and ΔG ^ο _rxn2 ≥0. However, since the relative concentrations of the blocker and primer can influence the equilibrium distribution and the effective thermodynamics of the reaction, the recommendations regarding ΔG ^ο _rxn1 and ΔG ^ο _rxn2 are not absolute.

In FIG. 3 shows in a schematic form the calculation of ΔG ^ο values based on the sequence. There are various agreements regarding the calculation of ΔG ^ο interactions of different sites; in FIG. Figure 3 shows approximate calculations of energy based on the model of nearest neighbors. The terms ΔG ^ο _1-5 , ΔG ^ο _4-7 and ΔG ^ο _4-8 , in addition, include the standard free hybridization initiation energy (ΔG ^ο _init ), as well as additional stacking adjacent to the sixth section. Standard free energies, ΔG ^ο calculated using other methods, can lead to the same values of ΔG ^ο _PT , ΔG ^ο _PV , ΔG ^ο _BT and ΔG ^ο _BV , although the thermodynamics of individual sections (for example, ΔG ^ο _3-6 ) may differ . Illustrative examples are intended to demonstrate the ΔG ^ο calculation method used in the present invention and are not intended to indicate sequences for analysis; the listed sequences may be too short to stably bind. The calculation of ΔG ^ο _PT , ΔG ^ο _PV , ΔG ^ο _BT and ΔG ^ο _BV based on the primer sequence, blocker sequence, target sequence, sequence variant, operating temperature and operating buffer conditions are known to those skilled in the art.

In FIG. 4 shows the probe and construction of the blocker of the present invention and the subsequence around the SMAD7 gene locus of two human deposited samples NA18537 and NA18562. The primer / blocker kit design is designed to specifically amplify the A variant of SMAD7 single nucleotide polymorphism (SNP). The blocker, in accordance with the concept, is fully complementary to the matrix variant carrying the G-variant SNP SMAD7 and is functionalized at the 3'-end using the 3-carbon linker (C3), which prevents the enzymatic extension using Taq DNA polymerase. The reverse primer binds to a sequence 3 ′ from the forward primer and is not specific to one or another allele. To explain the terminology used to describe the various subsequences of the primer and blocker, oligonucleotides can be described as follows. An oligonucleotide as a blocker includes a target-neutral subsequence, which includes a sequence located 5 'and 3' from the cytosine nucleotide, which is presented out of combination with the rest of the sequence - the cytosine nucleotide in this case is a blocking variable subsequence of the blocker. The overlapping subsequence of the oligonucleotide as a primer includes a sequence that is homologous to the blocker sequence on the 5 ′ side of the blocking variable subsequence (cytosine).

In FIG. 5 demonstrated efficacy pair primer-blocker, which is a target SNP rs3789806 (C / G) C - allele of a human, at a temperature of annealing / elongation in the range from 56 to 66 ^ο C ^ο C.

In FIG. 6A and FIG. 6B shows primer designs with a large design field to avoid unwanted interactions.

In FIG. 7A and FIG. 7B demonstrates the control of the behavior of allele-specific PCR by calculating ΔG ^ο _rxn1 . The simulation shown in FIG. 7A, suggest that when the primer and blocker are designed such that ΔG ^ο _rxn1 is more positive, the values of ΔCq are greater. However, with more positive values of ΔG ^ο _{rxn1, the} amplification of the target also slows down, which leads to a larger value of Cq. Modeling assumes a fixed value ΔΔG ^ο = 2 kcal / mol. The upper subgraphs show the predicted effects of various values of ΔG ^ο _rxn1 (asterisks) and ΔG ^ο _rxn2 (dots) on the outputs of the hybridization reaction. The lower subgraphs show the curve of the simulated target amplification and variant for each pair of primer and blocker. In all cases presented, the ratio of blocker to primer concentrations is 20: 1. In FIG. 7B (I) and FIG. 7B (II) presents the experimental results of the effects of ΔG ^ο _rxn1 on the selectivity of amplification. Different values of ΔG ^ο _rxn1 were achieved by lengthening or shortening the 3ʹ-end of the blocker. As expected, a more positive value of ΔG ^ο _rxn1 leads to a larger value of Cq for both the target and the variant. The authors of the present invention noted the optimal intermediate ΔG ^ο _rxn1 , leading to the largest value of ΔCq and a relatively small gap Cq (Cq <28) for the target. In contrast to the simulation, ΔCq did not increase monotonously with ΔG ^ο _rxn1 . The formation of primer dimers and nonspecific amplification gives rise to a limit level of Cq.

Kinetic modeling of the PCR process is presented as follows:

Tfn + 1 = Tfn + Pn • [Pn / (Pn + Bn) + Y (ΔG ° _rxn1 ) • Bn / (Pn + Bn)] • Trn

Trn + 1 = Trn + Rn • Tfn

Vfn + 1 = Vfn + Pn • [Pn / (Pn + Bn) + Y (ΔG ° _rxn2 ) • Bn / (Pn + Bn)] • Vrn

Vrn + 1 = Vrn + Rn • Vfn

where Tfn represents the normalized concentration of the target forward chain in cycle n, Trn represents the normalized concentration of the reverse chain of the target in cycle n, Vfn represents the normalized concentration of the forward chain of the variant in cycle n, Vrn represents the normalized concentration of the reverse chain of the variant in cycle n, Pn represents the normalized concentration of the forward primer in cycle n, Bn represents the normalized concentration of the blocker in cycle n, Rn represents the normalized concentration back of primer in a cycle n, and Y (ΔG ^ο) is the output of the hybridization reaction in a substitution reaction with the standard free energy ΔG ^ο reaction. The simulation results are presented in FIG. 7A.

In FIG. Figure 8 shows a blocker with a non-homologous sequence at the 3'-end, and not with non-extendable chemical functionalization. Since non-homologous sequences do not bind to the 3'-region of either the target or variant, the non-homologous region effectively prevents enzymatic elongation. The Cq and ΔCq values for the target are similar to those shown in Example 1, which indicates that, like functional groups such as a 3-carbohydrate spacer or dideoxynucleotide, a non-homologous sequence at the 3'-end can effectively prevent enzymatic extension. Here, fP, rP, and B are the forward primer, reverse primer, and blocker (for the forward primer), respectively.

With standard PCR blocking, only the forward primer is configured to amplify the target sequence; the reverse primer amplifies both the target and the variant approximately equal. The specificity of amplification can be further increased (with approximately a quadratic increase in sensitivity to mutations) if both the forward and reverse primers were configured to amplify only the target sequence. In order to achieve double target-specific amplification, the forward and reverse primers should be separated by a small distance as planned, so that they overlap with the variant-specific blocker. In the limit, the primer binding sites are separated by one nucleotide, the name of which differs between the target and the variant. The forward and reverse variant-specific blockers are partially complementary to each other since they bind to the complementary chains of the target and both span the variation site. Blockers are designed so that, despite their partial complementarity, under operating conditions, most blocker molecules do not hybridize with each other.

In each of FIG. 9A and FIG. 9B shows that primer-blocker systems designed for human SNP BRAF rs3789806 and IL23R RS 1884444 were multiplexed in the same reaction. TaqMan® probes, aimed at the appropriate sequence located 3ʹ from the binding sites of the blocker, were developed and used as a reading mechanism.

In FIG. 10 shows that non-specific binding of a primer or blocker to other primers, blockers or matrices results in non-specific amplification. The bottom panel represents a tread structure that inhibits non-specific binding of primer and blocker. Oligonucleotides as protectors are partially complementary to the primer and blocker sequences. The presence of a stoichiometric excess of treads results in the fact that both the primer and the blocker are partially double-stranded. The blocker has a new section 11, the sequence of which is not complementary to section 5 in the nucleic acid sequence, and the protector has a new section 10, the sequence of which is complementary to section 11.

In each of FIG. 11A and FIG. 11B shows primer and blocker designs for detecting deletion and insertion. The primer is not target-specific; this method can also be used to detect deletions or insertions with an indefinite number of bases or an indeterminate position.

In FIG. 12 shows the worst implementation of the present invention by placing primer 3 ′ from the SNP position and blocker 5 ′ from the primer. This implementation creates the specificity of hybridization only in the first cycle, therefore, ΔCq is less than in the case of the preferred design.

In FIG. 13 shows the selective amplification of subsequences of 18S fungal DNA from 3 pathogenic fungal species in a large excess of human homologous DNA. Since the subsequences of 18S fungi and the human subsequence homologous to them differ in many regions, the authors of the present invention have developed appropriate types of blocker for both the forward primer and the reverse primer to maximize amplification specificity.

In FIG. Figure 14 shows the use of target-specific amplification for both the forward and reverse primers using two sets of blockers specific for the allele variant, one for each primer. Blockers are partially complementary to each other, but under operating conditions they do not predominantly hybridize with each other. Blockers and primers are designed so that the forward primer cannot extend from the reverse blocker, and the reverse primer cannot extend from the forward blocker. In FIG. 14 presents preliminary experimental results.

Combinations of primer and blocker can be applied to other enzymatic amplification assays of nucleic acids, including, but not limited to, amplification reactions using single stranded break enzyme (NEAR), loop isothermal amplification (LAMP) and rolling ring amplification (RCA) .

The present invention is particularly suitable for detecting a small amount of a target in a large excess of variant. For the cancer diagnostic applications of the present invention, the variant may relate to a wild-type DNA sequence, and the target may relate to a cancerous DNA sequence. For applications in the form of the diagnosis of infectious diseases of the present invention, the variant may relate to the DNA sequence of the pathogen, and the target may relate to a homologous sequence of human DNA.

Examples

Example 1

In FIG. 4 illustrates Example 1 of the present invention for a probe and blocker and subsequence design around the SMAD7 gene locus of two human deposited samples NA18537 and NA18562. Single nucleotide polymorphisms (SNPs) of rs4939827 (C / T) at the SMAD7 gene locus in healthy individuals were selected to verify the validity of the concept. The T allele as a target and the C allele as an option were arbitrarily determined. Samples of the deposited human genomic DNA NA18562 (homozygous for the C allele) and NA18537 (homozygous for the T allele) were purchased from Coriell® and the corresponding nucleotide sequences were obtained from the 1000 Genomes website. To prevent unwanted polymerization, the blocker was modified with a C3 spacer at the 3'-end. The experimental results of quantitative PCR (qPCR) distinguished 0.1% target (99.9% variant) from 0% target (100% variant). A sample containing 0.1% of the target was prepared by mixing 0.1% NA18537 with 99.9% NA18562. The average difference in the quantitative estimate of the cycle (ΔCq) between the 100% variant and the 100% target was 14.8, and between the 100% variant (denoted as “WT” in the graph) and 0.1% target was 4.2. All experiments were performed in a 96-well plate in a Bio-Rad CFX96 ™ qPCR device. In each well, 200 nM primer, 2 μM blocker and 20 ng human genomic DNA were mixed in Bio-Rad iTaq ™ SYBR® Green Supermix. After initiation of the process for 3 min at 95 ^ο C was performed 65 cycles, each cycle consisting of a denaturation step at 95 ^ο C for 10 seconds and annealing step / ^ο elongation at 60 C for 30 seconds. In the experimental examples shown in FIG. 5-12, used a similar protocol.

Table 1

Gene SNP Blocker Cq digits NA18537 NA18562 ΔCq Braf rs3789806
G or C - 23,2 23,4 0.2 C 38.5 25.3 13,2 G 30.1 42.7 12.6 Egf rs11568849
C or A - 22.5 22.3 0.2 A 34.7 26.3 8.4 C 24.7 39.33 14.6 IL23R rs1884444
G or T - 22.2 22.4 0.2 T 36.1 24 12.1 G 28.3 35,4 7.1 Alk rs2246745
A or T - 22.6 22.3 0.3 A 23,2 33 9.8 T 33,2 24.1 9.1

Table 1 shows the results of allele-specific amplifications for four sets of SNP pairs in the NA18562 and NA18537 human genomic DNA samples taken from the 1000 Genomes database. For each pair of SNPs, primer and blocker designs for each variant, alleles have been developed and tested. The base names indicated in the “Blocker” column indicate the allele that was suppressed (option), and “-” means that no blocker was added. In all cases, large ΔCq values were noted between two alleles, in the range from 7.1 to 14.6. All experiments were performed in triplicate using 400 nM of each primer and 4 μM blocker in a Bio-Rad iTaq ™ SYBR® Green Supermix. The effectiveness of thermodynamically controlled designs without any empirical optimization of primer and blocker sequences or experimental conditions has been demonstrated using the results.

Example 2

In FIG. 5 shows Example 2 of the present invention for primer pair efficiency-blocker, which is a target SNP rs3789806 (C / G) C - allele of a human, at a temperature of annealing / elongation in the range from 56 to 66 ^ο C ^ο C. Example 2 showed good allele -specific amplification from 56 ^ο C to 66 ^ο C and have the stability to temperature deviation, is at least 8 ^ο C. was used 400 nM of each primer and 4 uM blocker, and other experimental conditions other than the temperature of annealing / elongation were same as described in the example 1.

Example 3

In FIG. 6A and FIG. 6B shows an example 3 of the present invention. The primer designs shown in the present invention provided a larger design field to avoid undesired interactions. Nucleic acid sequences of four sets of primer and blocker pairs, each of which specifically amplifies the SNP rs3789806 C allele in humans, demonstrated ΔCq> 11. 400 nM of each primer and 4 μM blocker were used, and other experimental conditions were the same as described in Example 1.

Example 4

In FIG. 7A, FIG. 7B (I) and FIG. 7B (II), Example 4 of the present invention is presented for controlling the behavior of an allele-specific PCR by calculating ΔG ^ο _rxn1 . The simulation shown in FIG. 7A suggested that when the primer and blocker were designed so that ΔG ^ο _rxn1 was more positive, the ΔCq values were larger. However, with more positive values of ΔG ^ο _{rxn1, the} amplification of the target also slowed down and led to a larger value of Cq. Modeling was allowed at a fixed value ΔΔG ^ο = 2 kcal / mol. The upper subgraphs predicted the effects of different values of ΔG ^ο _rxn1 (asterisks) and ΔG ^ο _rxn2 (dots) on the outputs of the hybridization reaction. The lower subgraphs predicted a curve of simulated amplification of the target and variant for each pair of primer and blocker. In all cases presented, the ratio of blocker to primer concentrations was 20: 1. In FIG. 7B (I) and FIG. 7B (II) presents the experimental results of the effects of ΔG ^ο _rxn1 on the selectivity of amplification. Different values of ΔG ^ο _rxn1 were achieved by lengthening or shortening the 3ʹ-end of the blocker. As expected, a more positive value of ΔG ^ο _rxn1 led to a larger value of Cq for both the target and the variant. The optimal intermediate ΔG ^ο _rxn1 led to the largest ΔCq value and a relatively small lag Cq (Cq <28) for the target. In contrast to the simulation, ΔCq did not increase monotonically with ΔG ^ο _rxn1 , since the formation of primer dimers and nonspecific amplification generated a limiting level of Cq. 200 nM of each primer and 2 μM blocker were used, and other experimental conditions were the same as described in Example 1.

Example 5

In FIG. 8 shows an example 5 of the present invention for a blocker with a non-homologous sequence at the 3'-end, and not with non-extendable chemical functionalization. Since non-homologous sequences do not bind to the 3'-region of either the target or variant, the non-homologous region effectively prevents enzymatic elongation. 400 nM of each primer and 4 μM blocker were used, and other experimental conditions were the same as described in Example 1. The values of Cq and ΔCq were similar to those shown in FIG. 4, which indicates that, like functional groups, such as a 3-carbohydrate spacer or dideoxynucleotide, a non-homologous sequence at the 3'-end effectively prevents enzymatic extension. In FIG. 8, fP, rP, and B are the forward primer, reverse primer, and blocker (for the forward primer), respectively.

Example 6

In FIG. 9A and FIG. 9B shows an example 6 of the present invention. To carry out real-time PCR using a non-allele-specific primer and an allele-specific blocker of the present invention, a reverse primer that was not allele-specific was also needed. SYBR® Green Supermix and TaqMan® were used in the experiments, and the results did not differ significantly between them. The primer-blocker systems designed for the SNP BRAF rs3789806 and IL23R RS 1884444 people were multiplexed in the same reaction. TaqMan® probes, aimed at the appropriate sequence located 3ʹ from the binding sites of the blocker, were developed and used as a reading mechanism. The TaqMan® probe for BRAF rs3789806 amplicons was modified using the FAM fluorophore, Iowa Black FQ quencher and internal ZEN quencher, and the TaqMan® probe for IL23R rs884444 amplicons was modified using Cy5 fluorophore and Iowa Black RQ quencher. In FIG. 9A shows the results of amplification - multiplexed detection of BRAF rs3789806 G-alleles and IL23R RSL 884444 T-alleles. The names of the bases indicated which allele is suppressed by blockers (option). In FIG. 9B shows the multiplexed detection of the BRAF rs3789806 C allele and IL23R RSL 884444 G allele. In each experiment, a genomic DNA sample was mixed with 400 nM of each primer, 4 μM of each blocker, and 200 nM of each TaqMan® probe in Bio-Rad iQ Supermix. The qPCR protocol was the same as indicated in Example 1.

Example 7

In FIG. 10 illustrates Example 7 of the present invention for nonspecific binding of a primer or blockers to other primers, blockers or matrices. Example 7 led to non-specific amplification. The bottom panel introduced a tread design that inhibits non-specific binding of primer and blocker. The blocker had a new region 11, the sequence of which was not complementary to region 5 in the nucleic acid sequence, and the tread had a new region 10, the sequence of which was complementary to region 11.

Example 8

In FIG. 11A and FIG. 11B shows an example 8 of the present invention for primer and blocker constructs for detecting deletion and insertion. Del rs200841330 was used to demonstrate the validation of the concept. 400 nM of each primer and 4 μM blocker were used, and other experimental conditions were the same as described in Example 1. Since the primer is not target-specific, this method can also be used to detect a deletion or insert with an indefinite number of bases or an undefined position.

Example 9

In FIG. 12 shows an example 9 of the present invention when placing the primer 3 ′ from the SNP position and the blocker 5 ′ from the primer. Example 9 created hybridization specificity only in the first cycle, therefore, ΔCq was less than in the case of the preferred design. The primer concentration was 400 nM and the blocker concentration was 4 μM. The experimental conditions were the same as indicated in example 1.

Example 10

In FIG. 13 illustrates Example 10 of the present invention for selectively amplifying subsequences of 18S fungal DNA from 3 pathogenic fungal species in a large excess of human homologous DNA. Since the subsequences of 18S fungi and the human subsequence homologous to them differ in many regions, the corresponding types of blocker were developed for both the forward primer and the reverse primer to maximize amplification specificity. The gBlock fragments (400 bp double-stranded DNA tested for IDT) from 18S subsequences of 3 species of fungi and a human consensus sequence were used in experiments. 60,000 copies (0.1%), 60 copies (0.0001%) and 0 copies (100% human) of each gBlock DNA fragment of the fungus were individually mixed with 60,000,000 copies of human DNA. In all cases, rare sequences were found up to 1 part in 1 million. Used 200 nm of each primer and 1 μm of each blocker, and other experimental conditions were the same as described in example 1.

Example 11

In FIG. 14 shows an example 11 of the present invention. In FIG. Figure 14 shows the use of allele-specific amplification for both forward and reverse primers; two sets of blockers specific to the allele variant were used, one for each primer. The blockers were partially complementary to each other, but under operating conditions they did not predominantly support hybridization with each other. Blockers and primers have been designed so that the forward primer cannot extend from the reverse blocker, and the reverse primer cannot extend from the forward blocker. In FIG. 14 presents preliminary experimental results.

The foregoing description of specific embodiments of the present invention has been presented for purposes of illustration and description. An exemplary embodiment has been selected and described to best explain the principles of the invention and its practical application, thereby making it possible for others skilled in the art to make the best use of the invention, and various embodiments with various modifications are suitable for the particular intended application.

Claims (66)

1. The composition of the oligonucleotides for amplification of the target sequence, including an oligonucleotide as a blocker comprising a functional group or a non-complementary portion of a sequence at or near the 3'-end that prevents enzymatic extension, where the oligonucleotide as a blocker includes a first sequence comprising a target-neutral subsequence and a blocking variable subsequence, where the blocker is variable the subsequence is flanked at its 3'- and 5'-ends by a neutral subsequence with respect to the target and inextricable with a neutral subsequence in relation to the target, an oligonucleotide as a first primer that is sufficient to induce enzymatic extension; where the oligonucleotide as the first primer comprises a second sequence, where the second sequence overlaps with a target neutral sequence of the oligonucleotide as a blocker for at least 5 nucleotides, so the second sequence includes an overlapping subsequence and non-overlapping subsequence, and where the second sequence does not include blocking variable subsequence, where the second sequence creates the standard free hybridization energy (ΔG ^ο _PT ), and the first sequence creates the standard free hybridization energy (ΔG ^ο _BT ), which satisfies the following condition: +2 kcal / mol≥ΔG ^ο _PT -ΔG ^ο _BT ≥ -8 kcal / mol where the concentration of the oligonucleotide as a blocker is about 2 to about 10,000 times higher than the concentration of the oligonucleotide as the first primer. 2. The oligonucleotide composition according to claim 1, wherein the functional group includes a 3-carbon spacer or dideoxynucleotide. 3. The oligonucleotide composition according to claim 1, wherein the overlapping sequence comprises a portion of the 5'-end of a subsequence neutral to the target, wherein said portion is from about 5 nucleotides to about 40 nucleotides. 4. The oligonucleotide composition according to claim 1, wherein the overlapping sequence comprises a portion of the 5'-end of a subsequence neutral to the target, wherein said portion is from about 7 nucleotides to about 30 nucleotides. 5. The oligonucleotide composition according to claim 1, where the non-overlapping subsequence creates a standard free hybridization energy (ΔG ^ο ₃ ), which satisfies the following condition: -4 kcal / mol≥ΔG ^ο ₃ ≥-12 kcal / mol 6. The oligonucleotide composition according to claim 1, wherein the concentration of the oligonucleotide as a blocker is about 5 to about 1000 times higher than the concentration of the oligonucleotide as the first primer. 7. The oligonucleotide composition according to claim 1, further comprising an oligonucleotide as a second primer, sufficient to induce enzymatic extension, where the oligonucleotide as a second primer comprises a third sequence, where the third sequence does not overlap with the first sequence or second sequence and where the third sequence is neutral in relation to the target and sufficient for use with the oligonucleotide as the first primer for amplification of the site of the nucleus oic acid using polymerase chain reaction. 8. The oligonucleotide composition according to claim 7, further comprising an oligonucleotide as a second blocker, comprising a second functional group or a second non-complementary portion of the sequence at or near the 3'-end, which prevents enzymatic extension, where the oligonucleotide as the second blocker includes a fourth sequence, comprising a second target-neutral subsequence and a second blocking variable subsequence, where the second blocking variable I subsequence is a sequence complementary to a blocking variable subsequence, where the second blocking variable subsequence is flanked at its 3'- and 5'-ends of the second target-neutral subsequence and is inextricable with the second target-neutral subsequence, where the third sequence overlaps with the second neutral to the target subsequence for at least 5 nucleotides, so the third the sequence includes a second overlapping subsequence and a second non-overlapping subsequence, and where the third sequence does not include a second blocking variable subsequence. 9. The oligonucleotide composition of claim 8, wherein the second functional group includes a 3-carbon spacer or dideoxynucleotide. 10. The oligonucleotide composition according to claim 8, wherein the second overlapping sequence comprises a portion of the 5'-end of the second subsequence neutral to the target, wherein said portion is from about 5 nucleotides to about 40 nucleotides. 11. The oligonucleotide composition according to claim 8, wherein the second overlapping sequence comprises a portion of the 5'-end of the second subsequence neutral to the target, wherein said portion is from about 7 nucleotides to about 30 nucleotides. 12. The oligonucleotide composition according to claim 8, where the third sequence creates the standard free hybridization energy (ΔG ^ο _PT2 ), and the fourth sequence creates the standard free hybridization energy (ΔG ^ο _BT2 ), which satisfies the following condition: +2 kcal / mol≥ΔG ^ο _{PT2 -ΔG} ^ο _BT2 ≥-8 kcal / mol 13. The oligonucleotide composition according to claim 8, where the second non-overlapping subsequence creates a standard free hybridization energy (ΔG ^ο ₆ ), which satisfies the following condition: -4 kcal / mol≥ΔG ^ο ₆ ≥-12 kcal / mol 14. The oligonucleotide composition according to claim 8, wherein the concentration of the oligonucleotide as the second blocker is about 2 to about 10,000 times higher than the concentration of the oligonucleotide as the second primer. 15. The oligonucleotide composition according to claim 8, wherein the concentration of the oligonucleotide as the second blocker is about 5 to about 1000 times higher than the concentration of the oligonucleotide as the second primer. 16. The composition of the oligonucleotides according to any one of paragraphs. 1-15, further comprising a reagent necessary for the polymerase chain reaction. 17. The oligonucleotide composition according to any one of paragraphs. 1-15, further comprising a plurality of nucleoside triphosphates. 18. The composition of the oligonucleotides according to any one of paragraphs. 1-15, further comprising DNA polymerase. 19. The composition of the oligonucleotides according to any one of paragraphs. 1-15, further comprising a reagent necessary for the polymerase chain reaction, a variety of nucleoside triphosphates, DNA polymerase. 20. The oligonucleotide composition according to any one of paragraphs. 1-15, where the blocking variable subsequence is a single nucleotide. 21. The combination for amplification of the target sequence, including an oligonucleotide as a blocker comprising a functional group or a non-complementary portion of a sequence at or near the 3'-end that prevents enzymatic extension, where the oligonucleotide as a blocker includes a first sequence comprising a target-neutral subsequence and a blocking variable subsequence, where the blocker is variable the subsequence is flanked at its 3'- and 5'-ends by a neutral subsequence with respect to the target and inextricable with a neutral subsequence in relation to the target, an oligonucleotide as a first primer that is sufficient to induce enzymatic extension; where the oligonucleotide as the first primer comprises a second sequence, where the second sequence overlaps with a target neutral sequence of the oligonucleotide as a blocker for at least 5 nucleotides, so the second sequence includes an overlapping subsequence and non-overlapping subsequence, and where the second sequence does not include blocking variable subsequence, where the second sequence creates the standard free hybridization energy (ΔG ^ο _PT ), and the first sequence creates the standard free hybridization energy (ΔG ^ο _BT ), which satisfies the following condition: +2 kcal / mol≥ΔG ^ο _PT -ΔG ^ο _BT ≥ -8 kcal / mol. 22. The oligonucleotide composition according to claim 21, where the functional group includes a 3-carbon spacer or dideoxynucleotide. 23. The oligonucleotide composition of claim 21, wherein the overlapping sequence comprises a portion of the 5'-end of a subsequence neutral to the target, wherein said portion is from about 5 nucleotides to about 40 nucleotides. 24. The oligonucleotide composition of claim 21, wherein the overlapping sequence comprises a portion of the 5'-end of a subsequence neutral to the target, wherein said portion is from about 7 nucleotides to about 30 nucleotides. 25. The oligonucleotide composition of claim 21, wherein the non-overlapping subsequence creates a standard free hybridization energy (ΔG ^ο ₃ ) that satisfies the following condition: -4 kcal / mol≥ΔG ^ο ₃ ≥-12 kcal / mol 26. The composition of the oligonucleotides according to any one of paragraphs. 21-25, where the blocking variable subsequence is a single nucleotide. 27. A method of amplifying a target sequence, comprising the steps of (a) obtaining a sample containing one or more copies of the first nucleic acid, including a variant sequence, and possibly containing at least one copy of a second nucleic acid, including a target sequence, where each of the target sequence and variant sequence includes a homologous subsequence and a variable subsequence, where the variable subsequence includes at least one nucleotide and where the variable subsequence is the sequence the target is a target-specific subsequence, and the variable subsequence of a variant sequence is not a target-specific subsequence; (b) introducing an oligonucleotide as a blocker in the sample, where the oligonucleotide as a blocker includes a first sequence comprising a target neutral sequence and a blocking variable subsequence, where a target neutral sequence is complementary to a part of a homologous subsequence and a blocking variable sequence is a subsequence being a target-specific subsequence, where the blocking variable sub-sequence sequence is flanked at its 3'-and 5'-ends relative to a neutral target subsequence and inseparable from neutral to a subsequence of the target; (c) introducing the oligonucleotide as the first primer into the sample, where the oligonucleotide as the first primer is sufficient to induce enzymatic extension, where the oligonucleotide as the first primer comprises a second sequence, where the second sequence is complementary to the second part of the homologous subsequence, where the second sequence overlaps with neutral to the target subsequence for at least 5 nucleotides, so the second lnost includes overlapping subsequence and non-overlapping subsequence, wherein the second sequence does not include any sequence complementary variable subsequence, wherein the second sequence creates a standard free energy of hybridization (ΔG ^ο _PT), a first sequence creates a standard free energy of hybridization (ΔG ^ο _BT), which satisfies the following condition: 2 kcal / mol≥ΔG ^ο _PT -ΔG ^ο _BT ≥-8 kcal / mol, wherein the concentration of an oligonucleotide as block ora in about 2 - about 10,000 times higher oligonucleotide concentrations as the first primer; (d) introducing into the sample DNA polymerase, nucleoside triphosphates and one or more reagents necessary for the polymerase amplification of nucleic acids; and (e) inducing a reaction in the sample under conditions sufficient to achieve amplification of nucleic acids. 28. The method according to p. 27, in which the oligonucleotide as a blocker includes a functional group or non-complementary portion of the sequence at the 3'-end or near it, which prevents enzymatic extension. 29. The method of claim 28, wherein the functional group comprises a 3-carbon spacer or dideoxynucleotide. 30. The method of claim 27, wherein the DNA polymerase is a thermostable DNA polymerase. 31. The method according to p. 30, in which the conditions sufficient to achieve amplification of nucleic acids include subjecting the sample to at least 10 cycles, where at least 2 different temperature effects are carried out in each cycle, one effect of a temperature of at least 85 ^ο С, and one influence of temperature, component not more than 75 ^ο С. 32. The method of claim 27, further comprising the step of introducing into the sample an enzyme selected from the group consisting of a single-stranded enzyme, recombinase, helicase, RNase, reverse transcriptase, or any combination thereof. 33. The method according to p. 27, in which the overlapping sequence includes a part of the 5'-end of a neutral sequence with respect to the target, where the specified part is from about 5 nucleotides to about 40 nucleotides. 34. The method according to p. 27, in which the overlapping sequence includes a part of the 5'-end of a neutral sequence with respect to the target, where the specified part is from about 7 nucleotides to about 30 nucleotides. 35. The method of claim 27, wherein the non-overlapping subsequence creates a standard free hybridization energy (ΔG ^ο ₃ ) that satisfies the following condition: -4 kcal / mol≥ΔG ^ο ₃ ≥-12 kcal / mol. 36. The method according to p. 27, in which the concentration of the oligonucleotide as a blocker introduced into the sample is about 5 to about 1000 times higher than the concentration of the oligonucleotide as the first primer introduced into the sample. 37. The method of claim 27, further comprising the step of introducing the oligonucleotide as the second primer into the sample, where the oligonucleotide as the second primer comprises a third sequence, where the third sequence does not overlap with the variable subsequence and where the third sequence is neutral with respect to the target and sufficient for use with an oligonucleotide as a first primer for amplification of a portion of a nucleic acid comprising a target sequence. 38. The method of claim 37, further comprising the step of introducing the oligonucleotide as a second blocker into the sample, wherein the oligonucleotide as a second blocker comprises a fourth sequence comprising a second target-neutral subsequence and a second blocker variable subsequence, where the second blocker variable subsequence is a sequence complementary to a blocking variable sub-sequence, where the second blocking variable sub-after The sequence is flanked at its 3'- and 5'-ends by a second target-neutral subsequence and is inextricable with a second target-neutral subsequence, where the third sequence overlaps with a second target-neutral subsequence for at least 5 nucleotides, so the third sequence includes a second overlapping subsequence and a second non-overlapping subsequence. 39. The method of claim 38, wherein the oligonucleotide as a second blocker comprises a second functional group or a second non-complementary portion of a sequence at or near the 3'-end that prevents enzymatic extension. 40. The method of claim 39, wherein the second functional group is selected from the group consisting of a 3-carbon spacer or dideoxynucleotide. 41. The method of claim 38, wherein the second overlapping sequence comprises a portion of the 5'-end of the second target-neutral subsequence, wherein said portion is from about 5 nucleotides to about 40 nucleotides. 42. The method of claim 38, wherein the second overlapping sequence comprises a portion of the 5'-end of the second target-neutral subsequence, wherein said portion is from about 7 nucleotides to about 30 nucleotides. 43. The method according to claim 38, in which the third sequence creates the standard free hybridization energy (ΔG ^ο _PT2 ), and the fourth sequence creates the standard free hybridization energy (ΔG ^ο _BT2 ), which satisfies the following condition: +2 kcal / mol≥ΔG ^ο _{PT2 -ΔG} ^ο _BT2 ≥-8 kcal / mol 44. The method of claim 38, wherein the second non-overlapping subsequence creates a standard free hybridization energy (ΔG ^ο ₆ ) that satisfies the following condition: -4 kcal / mol≥ΔG ^ο ₆ ≥-12 kcal / mol 45. The method of claim 38, wherein the concentration of the oligonucleotide as the second blocker introduced into the sample is about 2 to about 10,000 times higher than the concentration of the oligonucleotide as the second primer introduced into the sample. 46. The method of claim 38, wherein the concentration of the oligonucleotide as the second blocker introduced into the sample is about 5 to about 1000 times higher than the concentration of the oligonucleotide as the second primer introduced into the sample. 47. The method according to p. 27, comprising, after stage (e), the step of extracting an aliquot from the sample and repeating steps (b) through (e) inclusive.

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